1. Field of the Invention
The present invention relates to analogs of glycoprotein hormones and their preparation and use. More specifically, the invention relates to disulfide bond crosslinked analogs of the glycoprotein hormones and their preparation and use.
2. Description of the Related Art
The glycoprotein hormones include the hormones chorionic gonadotropin (CG) also known as choriogonadotropin, luteinizing hormone (LH) also known as lutropin, follicle stimulating hormone (FSH) also known as follitropin, and thyroid stimulating hormone (TSH) also known as thyrotropin. Those from humans are known as human chorionic gonadotropin (hCG), human luteinizing hormone (hLH), human follicle stimulating hormone (hFSH), and human thyroid stimulating hormone (hTSH). These hormones have important roles in gonadal and thyroid function (Pierce et al, 1981, Moyle et al, 1995). CG and LH bind to and stimulate LH receptors, FSH binds to and stimulates FSH receptors, and TSH binds to and stimulates TSH receptors. CG is a hormone produced in large quantities primarily by the placentas of a few mammals including those of primates. The amino acid sequences of the β-subunits of CG from primates usually differ from those of LH. Equines also produce a CG, however, this has the same amino acid sequence as equine LH (Murphy et al, 1991).
As reviewed by Pierce et al (1981), the glycoprotein hormones are heterodimers consisting of an α- and a β-subunit. The heterodimers are not covalently linked together and their subunits can be dissociated by treating the hormones with acid or urea (Pierce et al, 1981). Most higher vertebrates contain only one gene that encodes the α-subunit (Fiddes et al, 1984); the same α-subunit normally combines with the β-subunits of LH, FSH, TSH, and, when present, CG. Nonetheless, post-translational protein processing, notably glycosylation (Baenziger et al, 1988), can contribute to differences in the compositions of the α-subunits of LH, FSH, TSH, and CG. Most of the amino acid sequence differences between the hormones reside in their hormone-specific β-subunits (Pierce et al, 1981). These are produced from separate genes (Fiddes et al, 1984, Bo et al, 1992).
With few exceptions (Blithe et al, 1991), the α-β-heterodimers have much more hormonal activity than either free subunit (Pierce et al, 1981). The naturally occurring α- and β-subunits form α,β-heterodimers much better than they form α,α-homodimers or -homodimers. Indeed, expression of hCG α-subunit and β-subunit genes together in mammalian cells leads to the formation of α-β-heterodimers, α-subunit monomers, and β-subunit monomers. Only trace amounts, if any, of the α,α-homodimer or -homodimer are made or secreted from the cells.
High-resolution X-ray crystal structures of human chorionic gonadotropin (hCG) have been reported by two laboratories (Lapthorn et al, 1994; Wu et al, 1994). These structures revealed that the original proposed disulfide bond patterns (Mise et al, 1980 and 1981) were incorrect and that the hormone is a member of the cysteine knot family of proteins (Sun et al, 1995). Since the relative locations of the cysteines in all glycoprotein hormones are similar, they are likely to have the cysteine knot architecture found in hCG.
The locations of the cysteine residues in the α-subunits of the vertebrate glycoprotein hormones are similar (FIGS. 1A and 1B). Using the hCG α-subunit as a model, it is seen that the cysteine knot is formed by the second, third, fifth, seventh, eighth, and ninth α-subunit cysteines. This creates three large α-subunit loops (FIGS. 1A and 1B). Loop 1 is the sequence of amino acids between the second and third cysteines; loop 2 is the sequence of amino acids between the fifth and seventh α-subunit cysteines; and loop 3 is the sequence of amino acids between the seventh and eighth cysteines. The locations of the cysteine residues in the β-subunits of the vertebrate glycoprotein hormones are similar (Pierce et al, 1981). Using the hCG β-subunit as a model, it is seen that the cysteine knot is formed by the first, fourth, fifth, sixth, eighth, and ninth cysteines. This creates three large β-subunit loops (FIGS. 2A and 2B). Loop 1 is the sequence of amino acids between the first and fourth cysteines; loop 2 is the sequence between the fifth and sixth cysteines; and loop 3 is the sequence between the sixth and eighth cysteines. By replacing portions of the α-subunit with homologous portions of another α-subunit or by replacing portions of the β-subunit with homologous portions of another β-subunit, it is possible to prepare functional chimeras of each glycoprotein hormone subunit (Campbell et al, 1991; Moyle et al, 1990; Moyle et al, 1994; Cosowsky et al, 1995; Moyle et al, 1995; Cosowsky et al, 1997). A general schematic of the structural elements in glycoprotein hormone subunits is presented in FIG. 1C, and the correspondence with human glycoprotein hormone subunits by amino acid residues is shown below in Table 1A.
TABLE 1ACorrespondence of Human Hormone Subunit Amino Acid Sequencesto Structural Elements Depicted in FIG. 1CC-term/N-termLoop 1Loop 2Loop 3Seat-BeltCys-Knotα1-6α11-27α33-59α61-81α85-92α10, 28-32,CGβ1-8CGβ10-33CGβ39-56CGβ58-87CGβ91-14560, 82-84LHβ1-8LHβ10-33LHβ39-56LHβ58-87LHβ91-endCGβ 9, 34-38,FSHβ1-2FSHβ 4-27FSHβ33-50FSHβ52-81FSHβ85-11157, 88-90TSHβ1TSHβ 3-26TSHβ32-51TSHβ53-82TSHβ86-endLHβ 9, 34-38,57, 88-90FSHβ 3, 28-32,51, 82-84TSHβ 2, 27-31,52, 83-85
In addition to its cysteine knot, the β-subunit also contains a sequence termed the seat-belt (Lapthorn et al, 1994) that is wrapped around the second α-subunit loop. The seat-belt begins at the ninth cysteine, the last residue in the β-subunit cysteine knot, and includes the tenth, eleventh, and twelfth cysteines. It is latched to the first β-subunit loop by a disulfide bond formed between cysteine twelve (i.e., at the carboxyl-terminal end of the seat-belt) and cysteine three (i.e., in the first β-subunit loop).
The seat-belt is a portion of the hCG β-subunit that has a significant (if not primary) influence on the ability of hCG to distinguish LH and FSH receptors (Campbell et al, 1991; Moyle et al, 1994). Replacement of all or parts of the hCG seat-belt amino acid sequence with the seat-belt sequence found in hFSH altered the receptor binding specificity of the resulting hormone analog. Normally, hCG binds LH receptors more than 1000-fold better than FSH or TSH receptors. However, analogs of hCG such as CF94-117 and CF101-109 in which hCG seat-belt residues 101-109 (i.e., Gly-Gly-Pro-Lys-Asp-His-Pro-Leu-Thr) (residues 101-109 of SEQ ID NO:2) are replaced with their hFSH counterparts (i.e., Thr-Val-Arg-Gly-Leu-Gly-Pro-Ser-Tyr) (residues 113-121 of SEQ ID NO:15) bound FSH receptors much better than hCG (Moyle et al, 1994). Further, by manipulating the composition of the seat-belt, it is possible to prepare analogs of hCG that have various degrees of LH and FSH activities (Moyle et al, 1994; Han et al, 1996). These have potential important therapeutic uses for enhancing fertility in males and females.
Most, but not all of the intrasubunit disulfide bonds of the glycoprotein hormones are essential for their biological activities. Studies in which individual cysteines have been replaced by other amino acids, notably alanine, have shown that all the disulfide bonds of the cysteine knots and the seat-belt are essential for folding of the heterodimer (Suganuma et al, 1989; Bedows et al, 1993; Furuhashi et al, 1994). The remaining disulfide bonds between human α-subunit cysteines 7-31 and 59-87 and between hCG β-subunit cysteines 23-72 are not essential for heterodimer formation or for hormone activity.
As yet there is no high-resolution crystal structure that describes the interaction of any glycoprotein hormone with its receptor. Several models have been built in an effort to describe the structure of the hormone receptor complex. Most of these are based on the crystal structures of hCG and ribonuclease inhibitor, a protein that may be similar in structure to the extracellular domains of the glycoprotein hormone receptors. Most efforts to identify hormone residues that contact the receptor have been based on the influence of chemical, enzymatic, or genetic mutations that lead to a reduction in receptor binding. Unfortunately, since reduction in binding could be caused by disruption of a specific contact or by a change in hormone conformation (Cosowsky et al, 1997), the effects of these changes are difficult, if not impossible to interpret. This has led to considerable disagreement in this field (Remy et al, 1996; Berger et al, 1996) and some investigators have concluded that it is not possible to determine the orientation of the hormone in the receptor complex (Blowmick et al, 1996).
Other approaches to determine the orientation of the hormone in the receptor complex rely on identifying regions of the hormone that do not contact the receptor. These remain exposed after the hormone has bound to the receptor and/or can be altered without disrupting hormone-receptor interactions. When these are mapped on the crystal structure of hCG (Lapthorn et al, 1994; Wu et al, 1994), it is possible to develop a hypothetical model of the way that hCG might interact with LH receptors (Moyle et al, 1995). This approach suggested that the hormone groove formed by the second α-subunit loop and the first and third β-subunit loops is involved in the primary receptor contact (Cosowsky et al, 1995). This would also explain why both subunits are needed for highest hormone-receptor binding (Pierce et al, 1981). The data that support this conclusion are discussed in the next several paragraphs. However, it should be noted that most, if not all other investigators in this field support a model in which the hormone is oriented very differently (Remy et al, 1996; Berger et al, 1996), even though these investigators were aware of the model just described (i.e., they cite the paper describing the model in which the primary receptor binding site was made by the hormone groove).
Many portions of hCG α-subunit that do not appear to contact the receptor can be replaced without disrupting binding to LH receptors. Some of these regions are clearly exposed in the hormone-receptor complex since they can also be recognized by monoclonal antibodies while hCG is bound to LH receptors (Moyle et al, 1990; Cosowsky et al, 1995; Moyle et al, 1995). For example, although the human and bovine α-subunits have very different amino acid sequences, heterodimers containing the bovine α-subunit and the hCG β-subunit bind rat and human LH receptors well (Cosowsky et al, 1997). These heterodimers are readily distinguished by most monoclonal antibodies that recognize epitopes on loops 1 and 3 of the α-subunit of hCG (Moyle et al, 1995). These observations show that the surfaces of human and bovine α-subunit loops 1 and 3 differ and suggest that this region of the hormone does not form key essential receptor contacts.
By comparing the abilities of monoclonal antibodies to recognize analogs of hCG in which parts of the α-subunit were derived from either the human or bovine proteins, it was possible to identify key α-subunit residues that participated in antibody binding (Moyle et al, 1995). Some monoclonal antibodies that recognize epitopes on the hCG α-subunit also bound to a fragment of the α-subunit that had been prepared by trypsin digestion and that lacked most of the second α-subunit loop (Lapthorn et al, 1994; Wu et al, 1994; Birken et al, 1986). This observation was also used to determine and/or confirm the binding sites of these antibodies (Moyle et al, 1995). Two monoclonal antibodies that recognize α-subunit epitopes and that are referred to as A105 and A407 (Moyle et al, 1995) bound to hCG when the hormone was complexed with LH receptors. Thus, the α-subunit residues recognized by these antibodies do not appear to contact the LH receptor (Moyle et al, 1995). The remainder of the α-subunit includes the second α-subunit loop and the C-terminus of the protein. Some of the residues in these highly conserved portions of the α-subunit may participate in receptor contacts.
Many portions of hCG β-subunit that do not appear to contact the LH receptor can also be replaced by mutagenesis without disrupting LH receptor binding. This includes hCG β-subunit loop 2. An analog of hCG in which residues of hCG β-subunit loop 2 were replaced with those normally found in hFSH β-subunit loop 2, termed CF39-58 (Campbell et al, 1991), was readily distinguished by monoclonal antibodies that recognized FSH but not hCG residues in β-subunit loop 2. This showed that the structure of the second subunit loop differed in this analog and in hCG. Nonetheless, this β-subunit analog combined with the α-subunit to form an α,β-heterodimer that interacted with LH receptors similar to hCG (Campbell et al, 1991). Thus, few, if any, residues in the second β-subunit loop of hCG appear to make essential direct LH receptor contacts. Similarly, the second β-subunit loop of hFSH does not appear to contact FSH receptors. Analogs of hCG β-subunit have been reported in which residues between the tenth β-subunit cysteine and the C-terminus or between residues 94-114 were replaced with the corresponding residues of hFSH. These analogs are named CF94-117 (Campbell et al, 1991) and CFC94-114 (Wang et al, 1994). Both bind to FSH receptors much better than to LH receptors even though they contain the entire sequence of hCG in the second β-subunit loop. Because the second β-subunit loop of hCG appears to have minimal influence on the ability of the hormone to bind to either LH or FSH receptors, it seems unlikely that it participates in essential high affinity contacts with the LH or FSH receptor. Further, the second β-subunit loop of hCG is located near the first and third α-subunit loops, a portion of the α-subunit that also does not appear to contact the receptor. Thus, it appears likely that the entire region of hCG that contains residues in the first and third α-subunit loops and the second β-subunit loop may not contact the receptor. As will be discussed later, it is thought that this portion of the hormone projects into a cavity created by the horseshoe shape of the extracellular domain of the receptor (Moyle et al, 1995).
Residues of the hCG β-subunit that do not participate in high affinity receptor contacts can also be recognized by monoclonal antibodies after the hormone has bound to LH receptors (Campbell et al, 1991; Moyle et al, 1990; Moyle et al, 1994; Cosowsky et al, 1995). These include portions of the hCG β-subunit found in loops 1 and 3 furthest from the α-subunit interface that can be recognized by antibodies such as B105, B108, B111, and B112. These antibodies bind hCG when it is complexed with LH receptors (Moyle et al, 1990; Cosowsky et al, 1995), demonstrating that these portions of the β-subunit loops 1 and 3 do not make essential receptor contacts. Other studies have shown that it is possible to remove residues between the N-terminus and the first cysteine and between the twelfth cysteine and the C-terminus of the hCG β-subunit without eliminating the ability of the hormone to bind to LH receptors (Huang et al, 1993). The remaining portion of the β-subunit includes the seat-belt. Few of these residues appear to make essential contacts needed for high affinity binding to LH or FSH receptors. For example, it was found that changing the residues in the seat-belt between the eleventh and twelfth cysteines had minimal, if any, effect on binding to LH receptors (Moyle et al, 1994). Changing residues between the tenth and eleventh cysteines had less than five-fold effect on FSH receptor binding (Moyle et al, 1994). Further, most residues in the seat-belt of equine LH (eLH) and equine CG (eCG) differ from FSH (Pierce et al, 1981; Murphy et al, 1991), yet these hormones bind well to the rat FSH receptor. Indeed, purified eLH binds at least 30% as well as hFSH to the rat FSH receptor in vitro (Moyle et al, 1994). The portions of the hCG β-subunit that remain unaccounted include the surfaces of loops 1 and 3 that are nearest the α-subunit interface. Thus, these are likely sites of the hormone that contact the receptor. Portions of the α-subunit can also be recognized by monoclonal antibodies when hCG is complexed with LH receptors. These include residues recognized by antibodies A105 and A407 (Moyle et al, 1995).
Other strategies for identifying regions of the glycoprotein hormones that interact with receptors involve the use of hormone analogs that recognize more than one receptor (Campbell et al, 1991; Moyle et al, 1994; Han et al, 1996; Campbell et al, 1997). Thus, it was possible to prepare analogs of hCG that bind to FSH and TSH receptors simply by changing the β-subunit seat-belt of hCG to that found in hFSH (Campbell et al, 1997). This analog contains no TSH-specific residues yet was capable of stimulating a TSH response to the same maximum level as TSH. By comparing the activities of several of these multifunctional analogs, it is possible to conclude that most seat-belt residues and nearly all of the second β-subunit loop are unlikely to form key essential high-affinity receptor contacts.
There are no reports of a crystal structure for any LH, FSH, or TSH receptor. However, the amino acid sequences of several glycoprotein hormone receptors are known (Moyle et al, 1994; McFarland et al, 1989; Loosfelt et al, 1989; Segaloff et al, 1990; Sprengel et al, 1990; Braun et al, 1991; Nagayama et al, 1991; Nagayama et al, 1989; Jia et al, 1991). These proteins appear to have extracellular, transmembrane, and intracellular domains. When expressed without the transmembrane or intracellular domains (Braun et al, 1991; Ji et al, 1991; Xie et al, 1990; Moyle et al, 1991), or in conjunction with other transmembrane domains (Moyle et al, 1991), the extracellular domain is seen to contribute most of the affinity of the receptor for its ligand. The extra-cellular domains of these proteins are members of the leucine-rich repeat family of proteins and the transmembrane domains appear to have seven hydrophobic helices that span the plasma membrane (McFarland et al, 1989). A crystal structure of ribonuclease inhibitor, a model structure of one leucine-rich repeat protein has been determined and shown to have a horseshoe shape (Kobe et al, 1993 and 1995). This finding suggested that the extracellular domains of the LH, FSH, and TSH receptors are horseshoe-shaped (Moyle et al, 1995). Portions of the extracellular domain of the LH and FSH receptors that control their hCG and hFSH binding specificity have been identified through the use of LH/FSH receptor chimeras (Moyle et al, 1994). By considering the portions of hCG most likely to contact the LH receptor and the portions of the LH receptor that are responsible for ligand binding specificity, a model was developed that explains the abilities of this hormone to interact with LH receptors and to elicit signal transduction (Moyle et al, 1995). In this model, the groove between the second α-subunit loop and the first and third β-subunit loops contacts the rim of the receptor extracellular domain near the middle of the horseshoe (Moyle et al, 1995). The remainder of the hormone projects into the space between the arms of the horseshoe. When the hormone binds to the receptor and projects into this space, it stabilizes a conformation of the extracellular domain needed for signal transduction. This is conducted to the transmembrane domain by specific contacts between the extra-cellular and transmembrane domains needed for proper receptor expression on the cell surface (Moyle et al, 1991). The model accounts for the abilities of the oligosaccharides to influence signal transduction (Moyle et al, 1975; Matzuk et al, 1989). However, while the model can explain these data, other models in which different parts of hCG interact with the receptors have been proposed (Jiang et al, 1995). Thus, it is still unclear as to how the glycoprotein hormones interact with their receptors.
This view of hormone receptor interaction also explains the inhibition of hCG binding by some monoclonal antibodies that recognize regions of hCG thought not to participate in key receptor contacts. As discussed earlier, these include the hormone surface formed by α-subunit loops 1 and 3 and β-subunit loop 2. In the model, these regions project into a space between the N- and C-terminal arms of the receptor extracellular domain. Binding of antibodies to these sites prevents the hormone from entering this space.
Similarities in the locations of the cysteines in glycoprotein hormones from most vertebrate species (Pierce et al, 1981) suggest that they will fold like hCG. The structures of the receptors for the glycoprotein hormones are also likely to be quite comparable due to similarities in the leucine-rich repeats of their extracellular domains and the large number of conserved residues in their transmembrane domains (Moyle et al, 1994; Braun et al, 1991; Nagayama et al, 1991). Thus, it seems likely that any model that successfully explains the interaction of hCG with LH receptors will also predict the abilities of the other glycoprotein hormones to interact with their receptors. One way that the seat-belt can influence the specificity of ligand-receptor interaction would be to alter the relative positions of the hormone subunits (Cosowsky et al, 1997). This would change the shape of the groove between the second α-subunit loop and the first and third β-subunit loops. The suggestion has been made that inhibitory elements in the hormone and the receptor are responsible for preventing inappropriate ligand-receptor interactions (Moyle et al, 1994). Therefore, the effect of the seat-belt would be to alter the shape of the hormone to reduce its ability to fit into the central portion of the horseshoe.
The glycoprotein hormones have several therapeutic uses. FSH is used to induce development of ovarian follicles in preparation for ovulation induction in females (Galway et al, 1990; Shoham et al, 1991; Gast, 1995; Olive, 1995). LH and hCG are also used to induce ovulation of follicles that have initiated development. FSH, LH, and hCG are used to induce testis function in males. While the existing hormones can be used to stimulate the functions of the male and female gonads and the thyroid gland, practical application of the hormones for this use requires that they be heterodimers. These can be isolated from the pituitary gland (i.e., LH and FSH), serum (equine chorionic gonadotropin), or urine from pregnant (hCG) or postmenopausal women (mixtures of hLH and hFSH). Active heterodimers can also be isolated from cultures of cells that express both the α- and β-subunits including some from tumors (Cole et al, 1981) or those that have been transfected with cDNA or genomic DNA that encode the α- and β-subunits (Reddy et al, 1985). Indeed, the latter are an important source of glycoprotein hormones that have therapeutic utility. Because the oligosaccharides of the glycoprotein hormones have been shown to influence their abilities to elicit signal transduction (Moyle et al, 1975; Matzuk et al, 1989), preparation and synthesis of active heterodimers is best carried out in eukaryotic cells. These cells are capable of adding high mannose oligosaccharides to oligosaccharides and, in some cases, processing them to give the complex oligosaccharides that are found in the natural hormones (Baenziger et al, 1988). Nonetheless, because eukaryotic cells can process glycoproteins differently, synthesis of glycoprotein hormones is often carried out in mammalian cell lines such as that derived from the Chinese hamster ovary (CHO). While the hormones can be made in non-mammalian eukaryotic cells, the potential antigenicity of the oligosaccharide chains limits their clinical use.
The heterodimeric hormones have also been used as immunogens to elicit antisera that can be used to limit fertility (Singh et al, 1989; Pal et al, 1990; Talwar et al, 1986; Talwar et al, 1992; Moudgal et al, 1971 and 1972; Moudgal, 1976; Ravindranath et al, 1990; Moudgal et al, 1978). Due to the essential roles of hCG in maintaining human pregnancy, development of an immune response to hCG would be useful as a means of contraception and a substantial effort has been made to devise an hCG-based contraceptive vaccine. However, in principle, antibodies to the hormones could also be used to promote fertility. For example, LH levels appear to be excessive in some women who have polycystic ovarian disease. Thus, development of a method that would reduce but not eliminate circulating LH activity would be beneficial in restoration of fertility.
Glycoprotein hormone metabolism is very poorly understood. The half-lives of the hormones are known to be influenced by their content of oligosaccharides (Baenziger et al, 1988), particularly their terminal sugar residues. The most stable hormones are those that have the highest content of sialic acid in this location (Murphy et al, 1991; Baenziger et al, 1992; Fiete et al, 1991; Smith et al, 1993; Rosa et al, 1984). Nonetheless, the oligosaccharides are not entirely responsible for the stability of the hormones since the free hormone subunits are known to have significantly shorter circulating half-lives even though they have the same oligosaccharides as the heterodimers (Wehmann et al, 1984; Kardana et al, 1991). Indeed, it has been proposed that the hormones may be inactivated by proteolysis that leads to subunit dissociation (Kardana et al, 1991; Birken et al, 1991; Cole et al, 1991; Cole et al, 1991; Cole et al, 1993). Nicked hCG dissociated into its inactive subunits much faster than hCG (Cole et al, 1993). Thus, it is expected that a procedure that can prevent or reduce subunit dissociation would potentiate hormone efficacy.
The glycoprotein hormone subunits dissociate rapidly in denaturing conditions that include heat-treatment, extremes of pH, and urea (Pierce et al, 1981; Cole et al, 1993). For this reason, most glycoprotein hormone preparations are stored as lyophilized powders. A procedure that leads to enhanced stability of the heterodimer may enable hormone preparations to be stored and distributed in aqueous solutions. This would eliminate the need for shipping of separate hormone diluent and the additional step of hormone reconstitution by the end user.
Several attempts have been made to stabilize the hormones by “crosslinking” their subunits. Chemical crosslinking methods have been used (Weare et al, 1979 and 1979); however, these lead to reduced activity. It is also possible to genetically fuse the β- and α-subunits together to produce a single chain hormone. This molecule is more stable than the heterodimer and has high biological activity (Sugahara et al, 1995); however, it is grossly dissimilar from the native molecule.
Another method of crosslinking proteins would be to tether them by means of a disulfide bond. This strategy occurs naturally to stabilize other proteins of the cysteine knot superfamily (Sun et al, 1995) and probably takes the place of the seat-belt. Furthermore, addition of disulfide bonds to proteins can enhance their stability, provided the addition of the disulfide bond does not increase the internal strain within the protein (Matthews, 1987; Matsumura et al, 1989). However, no efforts have been reported to stabilize the glycoprotein heterodimers by a disulfide bond although a comment has been made that suggests the introduction of a disulfide bond may decrease their activity (Han et al, 1996). This is most likely because these heterodimers are complex proteins having several disulfides. Addition of a cysteine to these has the potential to disrupt folding, resulting in non-functional proteins. In addition, it is not likely that one can use other cysteine knot proteins as a basis to predict the locations of disulfide bonds that would stabilize the glycoprotein hormones as the organization of the subunits in the other cysteine knot proteins is substantially different from that in the glycoprotein hormones (Sun et al, 1995).
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